West nile vaccine

ABSTRACT

The present invention provides a safe and effective vaccine composition against West Nile virus disease. An immunogenically active component of West Nile virus or plasmid DNA, an adjuvant such as a metabolizable oil, and a pharmacologically acceptable carrier are formulated into an immunizing vaccine. The invention also provides a method for the prevention or amelioration of West Nile disease, such as encephalitis, in equidae by administering the vaccine composition herein set forth.

[0001] This application claims priority from co-pending U.S. applicationserial No. 60/308,334 filed on Jul. 27, 2001.

FIELD OF THE INVENTION

[0002] The present invention relates to safe and effective West NileVirus vaccines, and to methods of administering same to mammals, inparticular horses.

BACKGROUND OF THE INVENTION

[0003] Known as a Flavivirus, the West Nile virus was first identifiedin 1937 in Africa and first found in North America in 1999. Migratorybirds are considered the primary means whereby infection is spreadwithin and between countries. The virus is transmitted by mosquitoesthat have acquired infection by feeding on viremic birds. The virus isthen amplified during periods of adult mosquito blood-feeding. Infectedmosquitos then transmit the virus to humans and animals upon feedingthereon.

[0004] West Nile virus is the causative agent for West Nile Virusdisease, particularly West Nile encephalitis, predominately in humans,other mammals and birds. The chief concern in both the United States andforeign countries is the lack of effective treatment for West Nile virusdisease. Anti-inflammatory drugs are used to combat swelling of centralnervous system tissues, but beyond that no medical intervention isavailable. Nor is there believed to be a suitable vaccine known toprevent the infection. To date, preventing contact with carriers appearsto be the only means of controlling the West Nile virus.

[0005] What is therefore needed in the art is to provide a safe andeffective equine West Nile virus vaccine composition. The vaccinecomposition should be sufficiently safened so as to be suitable foradministration even to pregnant mares without adverse effect.

[0006] Also needed is a method for the prevention or amelioration ofWest Nile Virus disease, particularly West Nile encephalitis, in equidaeand other mammals.

SUMMARY OF THE INVENTION

[0007] The present invention provides a safened vaccine compositionwhich comprises: an effective immunizing amount of an immunogenicallyactive component selected from the group consisting of an inactivatedwhole or subunit West Nile virus, an antigen derived from said virus,DNA derived from said virus, and a mixture thereof; an immunogenicallystimulating amount of a metabolizable oil; and a pharmacologicallyacceptable carrier.

[0008] The present invention also provides a method for the preventionor amelioration of West Nile encephalitis in equidae which comprisesadministering to said equidae a safened vaccine composition whichcomprises an effective immunizing amount of an immunogenically activecomponent selected from the group consisting of an inactivated whole orsubunit West Nile virus, an antigen derived from said virus, DNA derivedfrom said virus, and a mixture thereof; an immunogenically stimulatingamount of a metabolizable oil; and a pharmacologically acceptablecarrier.

[0009] Also provided as part of the invention is a safe and effectiveWest Nile Virus vaccine suitable for use in horses which comprises animmunogenically active component selected from the group consisting ofan inactivated whole or subunit West Nile virus, an antigen derived fromsaid virus, DNA derived from said virus, and a mixture thereof; animmunogenically stimulating amount of a metabolizable oil; and apharmacologically acceptable carrier.

[0010] In a further embodiment, the invention describes a vaccinecomposition comprising at least about at least about 1×10⁴ TCID₅₀ perunit dose of inactivated West Nile virus, or a component thereof, andabout 4% to 10% vol/vol of a metabolizable oil comprising about 1 to 3%polyoxyethylene-polyoxypropylene block copolymer, about 2 to 6% ofsqualane and about 0.1 to 0.5% of polyoxyethylene sorbitan monooleate.

[0011] Further provided is a safened and effective West Nile virusvaccine for equidae, comprising at least about 1×10⁶ TCID₅₀ per unitdose of killed or inactivated West Nile virus, and at least about 1%vol/vol of an adjuvant comprising at least one metabolizable oil and atleast one wetting or dispersing agent.

[0012] The invention also sets forth a vaccine regimen for horses,comprising two dosage units of killed or inactivated West Nile virus,wherein each said dosage unit comprises about 0.5 to 5 milliliters of acomposition containing at least about 5×10⁷ TCID₅₀ of said virus andabout 1 to 10% vol/vol of an adjuvant, said adjuvant comprising at leastone metabolizable oil and at least two nonionic surfactants, and furtherwherein said dosage unit comprises a pharmacologically acceptablecarrier.

[0013] Also provided as part of the invention is an equine vaccinecontaining West Nile plasmid DNA in an amount of about 50 to 3,000micrograms per dose, together with one or more adjuvants and a suitablecarrier. A vaccine regimen would comprise administering at least aboutone dose of this composition, and desirably at least about 2 doses, tohorses for immunization against West Nile virus disease.

[0014] Further, objects and features of the invention will becomeapparent from the detailed description and the claims set forth hereinbelow.

DETAILED DESCRIPTION OF THE INVENTION

[0015] Scientists believe that the West Nile virus follows the samepattern of infection found with other mosquito-transmitted viruses. Whenan infected mosquito bites an equid, the virus enters the skin ortissues just below the bite site, where it is picked up by thecirculation. The virus can multiply in the bloodstream, and the equidmay develop a fever, which often goes undetected because there are noother signs of illness at the time. However, once the virus has invadedthe nervous system, clinical signs appear within one to three days. Mostaffected equidae, such as horses, first exhibit signs of posteriorweakness or paralysis and poor coordination. Depression and relatedbehavior changes may accompany the physical changes. In severe cases,tremors, convulsions, paddling of the limbs and paralysis may develop.Severe neurological problems and mortality have also been observed. Todate, no vaccine known to prevent West Nile virus in equidae isavailable; and the only means of controlling the West Nile virus appearsto be the prevention of contact with a carrier.

[0016] It has now been found that a safe and effective vaccinecomposition which comprises an effective immunizing amount of animmunogenically active component selected from the group consisting ofan inactivated whole or subunit West Nile virus, an antigen derived fromsaid virus, DNA derived from said virus, plasmid West Nile virus DNA,plasmid with sequence inserts of said virus, and a mixture thereof; animmunogenically stimulating amount of an adjuvant, in particular ametabolizable oil; and a pharmacologically acceptable carrier may beadministered to equidae, particularly horses, to prevent or ameliorateWest Nile Virus disease such as encephalitis.

[0017] DNA derived from the West Nile virus may be obtained viaisolation from sources such as the fluids or tissues of equine or avianspecies diagnosed to have West Nile encephalitis. Such sources includecerebral spinal fluid or sections of spinal cord or brain. DNA may alsobe obtained using other available techniques such as plasmid technology.For example, suitable cells of an organism, e.g. E. coli, may betransformed with a plasmid containing West Nile protein sequence insertsto obtain a master seed. The master seed may then be cultured andpassaged. Transformed cells containing the West Nile DNA may then beharvested, and the DNA isolated and obtained using techniques availableto the skilled artisan.

[0018] Whole or subunit West Nile virus may be isolated from infectedanimals using conventional techniques. Samples of the virus may also beobtained from tissue culture collections which maintain a depository fororganisms such as West Nile. At the American Type Culture Collection(ATCC), for example, the West Nile virus has been deposited under ATCCNo.s VR-82, VR-1267 and VR-1267 AF.

[0019] Whole or subunit West Nile virus may be inactivated byconventional inactivating means, for example chemical inactivation usingchemical inactivating agents such as binary ethyleneimine,beta-propiolactone, formalin, gluteraldehyde, sodium dodecyl sulfate, orthe like or a mixture thereof, preferably formalin. Said virus may alsobe inactivated by heat or psoralen in the presence of ultraviolet light.(Live, attenuated West Nile virus may also be used in certainembodiments, but this is perhaps much less preferred.)

[0020] Whole or subunit West Nile virus may be maintained or grown inmouse brains or in suitable tissue culture media, such as optiMEM (LTI,Grand Island, N.Y.) or MEM media, or in cells known in the art such asAfrican green monkey kidney (Vero) cells or baby hamster (BHK) cells,preferably Vero cells. Said virus may then be separated from the tissueculture or cell media using conventional techniques such ascentrifugation, filtration or the like.

[0021] A preferred West Nile virus isolate may be obtained from theNational Veterinary Services Laboratory (part of the United StatesDepartment of Agriculture) in Ames, Iowa as strain VM-2. The virusstrain may be plaque purified up to three times, and passaged to X+5 inVero cells.

[0022] As used herein the term “immunogenically active” designates theability to stimulate an immune response, i.e., to stimulate theproduction of antibodies, particularly humoral antibodies, or tostimulate a cell-mediated response. For example, the ability tostimulate the production of circulating or secretory antibodies or theproduction of a cell-mediated response in local mucosal regions, (e.g.,intestinal mucosa), peripheral blood, cerebral spinal fluid or the like.

[0023] The effective immunizing amount of the immunogenically activecomponent may vary and may be any amount sufficient to evoke an immuneresponse and provide immunological protection against West Nile Virusdisease. Amounts wherein a dosage unit comprises at least about 1×10⁴TCID₆₀ of killed or inactivated whole or subunit virus cells or antigenor DNA cells derived therefrom or a mixture thereof, preferably at leastabout 1×10⁶ TCID₅₀, are suitable. Even more preferably, at least about1×10⁷ TCID₅₀ per dosage unit may be utilized. It is especially desirablethat at least about 5×10⁷ TCID₅₀ of killed or inactivated whole orsubunit West Nile virus cells or antigen or DNA cells derived therefromor a mixture thereof be used in the vaccine composition of theinvention. In certain embodiments, as much as 1×10⁹ TCID₅₀ and more maybe utilized. A range of about 1×10⁴ TCID₅₀ to about 1×10⁸ TCID₅₀ mayalso be utilized.

[0024] In a further embodiment of the invention, it is contemplated thatabout 50 to 3,000 micrograms (μg or 10⁻⁶ grams) of West Nile plasmid DNAmay be utilized in one dosage unit of the vaccine composition. Morepreferably, about 100 to 1,000 μg may be used, with about 100 to 250 μgof plasmid DNA being more preferred.

[0025] At least one dosage unit per animal is contemplated herein as avaccination regimen. In some embodiments, two or more dosage units maybe especially useful. A dosage unit may typically be about 0.1 to 10milliliters of vaccine composition, preferably about 0.5 to 5milliliters, and even more preferably about 1 to 2 milliliters, witheach dosage unit containing the heretofore described quantity of virusor virus component. The skilled artisan will quickly recognize that aparticular quantity of vaccine composition per dosage unit, as well asthe total number of dosage units per vaccination regimen, may beoptimized, so long as an effective immunizing amount of the virus or acomponent thereof is ultimately delivered to the animal.

[0026] The West Nile virus vaccine composition of the invention may alsocontain one or more adjuvants. As used herein the term “adjuvant” refersto any component which improves the body's response to a vaccine. Theadjuvant will typically comprise about 0.1 to 50% vol/vol of the vaccineformulation of the invention, more preferably about 1 to 50% of thevaccine, and even more desirably about 1 to 20% thereof. Amounts ofabout 4 to 10% may be even more preferred.

[0027] Suitable adjuvants can include immunostimulating oils such ascertain metabolizable oils. Metabolizable oils suitable for use in thecomposition of the invention include oil emulsions, e.g., SP oil(hereinafter described), Emulsigen (MPV Laboratories, Ralston, NZ),Montanide 264,266,26(Seppic SA, Paris, France), as well as peanut oiland other vegetable-based oils, squalane (shark liver oil) or othermetabolizable oil which can be shown to be suitable as an adjuvant inveterinary vaccine practice.

[0028] In addition, the adjuvant may include one or more wetting ordispersing agents in amounts of about 0.1 to 25%, more preferably about1 to 10%, and even more preferably about 1 to 3% by volume of theadjuvant. Particularly preferred as wetting or dispersing agents arenon-ionic surfactants. Useful non-ionic surfactants includepolyoxyethylene/polyoxypropylene block copolymers, especially thosemarketed under the trademark PLURONIC® and available from BASFCorporation (Mt. Olive, N.J.). Other useful nonionic surfactants includepolyoxyethylene esters such as polyoxyethylene sorbitan monooleate,available under the trademark TWEEN 80®. It may be desirable to includemore than one, e.g. at least two, wetting or dispersing agents in theadjuvant as part of the vaccine composition of the invention.

[0029] Other components of the adjuvant may include such preservativecompounds as formalin and thimerosal in amounts of up to about 1%vol/vol of the adjuvant.

[0030] As an adjuvant, SP oil is preferred. As used in the specificationand claims, the term “SP oil” designates an oil emulsion comprising apolyoxyethylene-polyoxypropylene block copolymer, squalane,polyoxyethylene sorbitan monooleate and a buffered salt solution. Ingeneral, the SP oil emulsion will comprise about 1 to 3% vol/vol ofblock copolymer, about 2 to 6% vol/vol of squalane, more particularlyabout 3 to 6% of squalane, and about 0.1 to 0.5% vol/vol ofpolyoxyethylene sorbitan monooleate, with the remainder being a bufferedsalt solution.

[0031] When utilized, immunogenically stimulating amounts of SP oil asadjuvant in the vaccine composition of the invention may vary accordingto the immunogenically active component, the degree of potentialinfectious exposure, method of administration of the vaccinecomposition, the age and size of the equid, or the like. In general,amounts of about 1% to 50% vol/vol, preferably about 4% to 10% vol/vol,and more preferably about 4% to 5% vol/vol of SP oil are suitable.

[0032] In general, it is believed that a live virus vaccine maypotentially lack sufficient safety in a given target host, and that akilled or inactivated virus vaccine may potentially lack the ability tostimulate a sufficiently effective immunologic response. Commonly, anadjuvant or immunogenically stimulating compound is used in combinationwith a killed or inactivated virus in a vaccine composition to obtainacceptable efficacy. However, safety to the target host is oftencompromised by the addition of an adjuvant. For example, pregnantanimals many times have been known to have a significantly higher rateof miscarriage after being administered a killed or inactivated virusvaccine that contains an adjuvant.

[0033] It has now been found that when a suitable adjuvant, e.g. ametabolizable oil preferably such as SP oil, is used in combination withan immunogenically active component as described hereinabove, theresultant West Nile vaccine composition is safened for use in equidae,particularly horses, even for use in pregnant mares, while demonstratingimportant efficacy as well. Thus, the invention achieves the concomitantgoals of effective immunization and safety, especially for pregnantanimals. This combination of active immunogen and adjuvant is unheraldedin the art.

[0034] Pharmacologically acceptable carriers suitable for use in thevaccine composition of the invention may be any conventional liquidcarrier suitable for veterinary pharmaceutical compositions, preferablya balanced salt solution or other water-based solution suitable for usein tissue culture media. Other available carriers may also be utilized.

[0035] Additional excipients available in the art may also be Includedin the vaccine composition according to the various embodimentsheretofore described. For example, pH modifiers may be utilized.

[0036] The components of the vaccine composition of the invention asheretofore described, including the carrier, may be combined togetherusing available techniques.

[0037] In addition to the immunogenically active component of West Nilevirus as described hereinabove as active ingredient, it is contemplatedthat the vaccine composition of the invention may also contain otheractive components such as an antipathogenic component directed againstrabies virus, Eastern equine encephalitis virus, Western equineencephalitis virus, Venezuelan equine encephalitis virus, equine herpesvirus such as EHV-1 or EHV-4, Ehrlichia risticii, Streptococcus equi,tetanus toxoid, equine influenza virus(EIV), or the like or acombination thereof. The quantities of one or more of these viruses maybe determined from efficacy literature in the art, or determined usingavailable techniques.

[0038] In one embodiment of the invention the immunogenically activecomponent of the invention may be incorporated into liposomes usingknown technology such as that described in Nature, 1974, 252, 252-254 orJournal of Immunology, 1978, 120, 1109-13. In another embodiment of theinvention, the immunogenically active component of the invention may beconjugated to suitable biological compounds such as polysaccharides,peptides, proteins, or the like, or a combination thereof.

[0039] In a preferred embodiment of the invention, the inventive vaccinecomposition may be formulated in dosage unit form as heretoforedescribed to facilitate administration and ensure uniformity of dosage.Formulation may be effected using available techniques, such as thoseapplicable to preparations of emulsions.

[0040] The inventive vaccine composition may be administeredparenterally, for example, intramuscularly, subcutaneously,intraperitoneally, intradermally or the like, preferablyintramuscularly; or said composition may be administered orally orintranasally.

[0041] Accordingly, the present invention also provides a method for theprevention or amelioration of West Nile encephalitis in equidae,preferably horses, which comprises administering to said equidae asafened vaccine composition as described hereinabove.

[0042] In actual practice, the vaccine composition of the invention isadministered parenterally, subcutaneously, orally, intranasally, or byother available means, preferably parenterally, more preferablyintramuscularly, in effective amounts according to a schedule which maybe determined by the time of anticipated potential exposure to a carrierof the West Nile virus. In this way, the treated animal may have time tobuild immunity prior to the natural exposure. By way of non-limitingexample, a typical treatment schedule or dosing regimen may includeparenteral administration, preferably intramuscular injection of onedosage unit, at least about 2-8 weeks prior to potential exposure. Atleast two administrations are preferred, for example one dosage unit atabout 8 weeks and a second dosage unit at about 3-5 weeks prior topotential exposure of the treated animal. As heretofore set forth, adosage unit will typically be within the range of about 0.1 to 10milliliters of vaccine composition containing the previously describedamounts of active and percentages of adjuvant and inactive(s) as setforth. A dosage unit within the range of about 0.5 to 5 milliliters isperhaps more preferred, with about 1 to 2 milliliter(s) beingparticularly preferred.

[0043] For a clearer understanding of the invention, the followingexamples are set forth below. These examples are merely illustrative andare not understood to limit the scope or underlying principles of theinvention in any way. Indeed, various modifications of the invention, inaddition to those shown and described herein, will become apparent tothose skilled in the art from the following examples and the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims.

EXAMPLE 1 Preparation of Vaccine

[0044] A/Formulation of SP Oil Ingredient Description VolumePolyoxyethylene-polyoxypropylene block copolymer 20.0 ml (Pluronic ®L121, BASF, Mt. Olive, NJ) Squalane (Kodak, Rochester, NY) 40.0 mlPolyoxyethylenesorbitan monooleate  3.2 ml (Tween ® 80, Sigma Chemical,St. Louis, MO) Buffered salt solution 936.8 ml  (D-V PBS Solution, Ca,Mg free)

[0045] The ingredients are mixed and homogenized until a stable mass oremulsion is formed. Prior to homogenization, the ingredients or mixturemay be autoclaved. The emulsion may be further sterilized by filtration.Formalin may be added up to a final concentration of 0.2%. Thimerosalmay be added to a final dilution of 1:10,000.

[0046] B/Vaccine Preparation

[0047] An equine isolate of West Nile virus, obtained from USDAfacilities in Ames, Iowa (Lot No. VM-2, Equine Origin, 1999 NorthAmerican isolate, second passage in VeroM cell culture), was cultivatedin multiple cultures of Vero cells in OptiMEM (LTI, Grand Island, N.Y.)tissue culture medium at 37° C. The harvests are titrated and theninactivated by means of addition of a 10% formalin solution to a finalconcentration of 0.1%. This is allowed to inactivate at 37° C. for aperiod of no less than 144 hours. Then, another addition of 0.1%formalin is added and incubated at 37° C. for another period of no lessthan 144 hours.

[0048] The vaccines are formulated by suspending the appropriate volumeof inactivated virus fluid in 1-20% by volume of SP oil per 1 mL dose.

EXAMPLE 2

[0049] Evaluation of Antibody Response to the Intramuscular Injection ofTest Vaccine

[0050] In this evaluation, horses are randomly divided into four groups:one group of twenty horses is administered test vaccine at a dose of1×10⁷ TCID₅₀ (Tissue Culture Infectious Dose); a second group of twentyhorses is administered test vaccine at a dose of 5×10⁷ TCID₅₀; a thirdgroup of five horses is administered test vaccine at a dose of 1×10⁸TCID₅₀; and a fourth group of eight horses are maintained asnon-vaccinated environmental controls. Treated horses are given a firstdose of vaccine according to the group to which they are assigned. Attwenty-one days following administration of the first dose, a seconddose of the same vaccine is administered. All horses are bled for serumat the time of administration of the first and second dose and at weeklyintervals through 28 days post second dose administration. ComponentConc./Dose Volume/mL Test Vaccine A West Nile virus-Inactivated 1 × 10⁷TCID₅₀ 0.0347 mL MEM¹ NA 0.9138 mL SP oil 5% 0.0500 mL Polymyxin B² 30.0mcg/mL 0.0003 mL Neomycin 30.0 mcg/mL 0.0003 mL Thimerosal (5%) 1:20,0000.0010 mL Test Vaccine B West Nile virus-Inactivated 5 × 10⁷ TCID₅₀0.1734 mL MEM NA 0.7752 mL SP oil 5% 0.0500 mL Polymyxin B² 30.0 mcg/mL0.0002 mL Neomycin³ 30.0 mcg/mL 0.0002 mL Thimerosal⁴ (5%) 1:20,0000.0010 mL Test Vaccine C West Nile virus-Inactivated 1 × 10⁸ TCID₅₀0.3467 mL MEM¹ NA 0.6019 mL SP oil 5% 0.0500 mL Polymyxin B² 30.0 mcg/mL0.0002 mL Neomycin³ 30.0 mcg/mL 0.0002 mL Thimerosal⁴ (5%) 1:20,0000.0010 mL

[0051] The serological data obtained is shown in Table I below, wherein:0 DPV 1 designates day zero, pre vaccination; and 14 DPV 2 designatesday 14, post vaccination. NR designates no results.

[0052] As can be seen from the data on Table I, treated horses from allgroups showed significant increases in antibodies to West Nile viruswhile the control horses maintained a non-detectable antibody level. Thelevel of response in the horses that received vaccine was independent ofthe level of antigen in the vaccine that they received. TABLE I TestDose Test #1 Test #2 Vaccine (TC1D₅₀) 0 DPV 1 14 DPV 2 0 DPV 1 14 DPV 2A 1 × 10⁷ <10 160 <10 80 A 1 × 10⁷ <10 20 <10 10 A 1 × 10⁷ <10 ≧320 <1040 A 1 × 10⁷ <10 80 <10 ≧320 A 1 × 10⁷ <10 80 <10 40 A 1 × 10⁷ <10 80<10 160 A 1 × 10⁷ <10 40 <10 40 A 1 × 10⁷ <10 80 <10 80 A 1 × 10⁷ <10160 <10 80 A 1 × 10⁷ <10 40 <10 10 A 1 × 10⁷ <10 40 <10 10 A 1 × 10⁷ <10160 <10 40 A 1 × 10⁷ <10 ≧320 <10 NR* A 1 × 10⁷ <10 ≧320 <10 NR  A 1 ×10⁷ <10 ≧320 <10 80 A 1 × 10⁷ <10 160 <10 20 A 1 × 10⁷ ≧20 ≧320 <10 160A 1 × 10⁷ <10 80 <10 80 A 1 × 10⁷ <10 80 <10 160 A 1 × 10⁷ <10 160 <10160 B 5 × 10⁷ <10 80 <10 40 B 5 × 10⁷ <10 20 <10 <10 B 5 × 10⁷ <10 ≧320<10 160 B 5 × 10⁷ <10 80 <10 ≧320 B 5 × 10⁷ <10 ≧320 <10 160 B 5 × 10⁷<10 80 <10 80 B 5 × 10⁷ <10 160 <10 ≧320 B 5 × 10⁷ <10 80 <10 40 B 5 ×10⁷ <10 160 <10 40 B 5 × 10⁷ <10 40 <10 80 B 5 × 10⁷ <10 20 <10 20 B 5 ×10⁷ <10 ≧320 <10 160 B 5 × 10⁷ <10 ≧40 <10 ≧320 B 5 × 10⁷ <10 80 <10 40B 5 × 10⁷ <10 80 <10 NR  B 5 × 10⁷ <20 ≧320 <10 80 B 5 × 10⁷ ≧20 160 <1080 B 5 × 10⁷ <10 ≧320 <10 160 B 5 × 10⁷ <10 ≧320 <10 ≧320 B 5 × 10⁷ <1080 <10 160 C 1 × 10⁸ <10 ≧320 <10 ≧320 C 1 × 10⁸ <10 ≧320 <10 160 C 1 ×10⁸ <10 160 <10 80 C 1 × 10⁸ <10 ≧320 <10 NR  C 1 × 10⁸ <10 40 <10 40Control 0 <10 <10 <10 <10 Control 0 <10 <10 <10 <10 Control 0 <10 <10<10 <10 Control 0 <10 <10 <10 NR  Control 0 <10 <10 <10 <10 Control 0<10 <10 <10 <10 Control 0 <10 <10 <10 <10 Control 0 <10 <10 <10 <10

EXAMPLE 3

[0053] Evaluation of Safety of Test Vaccine in Horses Under FieldConditions

[0054] In this evaluation, 648 healthy male and female horses, including32 pregnant mares, are vaccinated with a 6×10⁶ TCID₅₀ dose ofinactivated test vaccine administered as a 1 mL dose vaccination byintramuscular administration and followed in three to four weeks by asecond 1 mL dose vaccination. The treated horses are housed and fedusing conventional husbandry practices for farm or stable. All treatedhorses are observed by a veterinarian for 30 minutes followingvaccination for immediate reactions such as salivation, labored orirregular breathing, shaking, or anaphylaxis. For two weekspost-vaccination, the horses are observed daily for any delayedreactions such as lethargy, anorexia or unusual swelling at theinjection site. Blood samples of 5 to 10 mL are taken by venipuncturefrom treated horses on the day of first vaccination (day Zero) and atleast once more at two or more weeks post second vaccination (day 36 orgreater). Serological assays using PRNT⁵ testing are performed. TestVaccine Component Conc./Dose Volume/mL West Nile virus-Inactivated 6 ×10⁶ TCID₅₀ 0.21 mL SP oil 5% 0.05 mL MEM N/A 0.74 mL

[0055] No vaccine-induced safety problems were found in any of thevaccinates, including the pregnant mares. This evaluation demonstratesthat the vaccine of the invention is safe for use in horses under fieldconditions.

EXAMPLE 4

[0056] Evaluation of Efficacy of Test Vaccine (Multivalent andMonovalent Preparations) in Horses Under Experimental Conditions

[0057] The efficacy of a combination vaccine containing killed West Nilevirus (WNV) against experimental WNV challenge was evaluated.

[0058] A total of 30 horses were allotted into one vaccinated group (20horses) and one control group (10 horses). Horses in the vaccinatedgroup received intramuscularly two doses of the test vaccine containingkilled West Nile virus (5×10⁷ TCID₅₀ per dose with 5% SP oil), influenzavirus, encephalomyelitis virus (Eastern, Western and Venezuelan),rhinopneumonitis virus (serotypes 1 and 4), and tetanus toxoid, threeweeks apart. Serum samples were collected periodically for antibodyresponse measured by plaque reduction neutralization test (PRNT).Twenty-four (24) days after the second vaccination, all horses werechallenged subcutaneously with WNV. After challenge, horses weremonitored for rectal temperature and any clinical signs twice daily fortwo weeks and once weekly thereafter for detection of viremia. Horseswere euthanized and necropsied on 21 and 22 DPC. Cerebrospinal fluid(CSF), spinal cord (cervical, thoracic, and lumbar) and brain (frontal,occipital, medulla oblongata, and brain stem) tissue samples wereexamined for gross pathology and collected for virus isolation.

[0059] Fourteen days after the second vaccination, 75% of vaccinatedanimals seroconverted (titer≧5) with a geometric mean titer of 10 whilecontrol animals remained negative (titer<5). The vaccination conferred asignificant protection against viremia (a precursor to development offull-blown West Nile Virus disease). Nine out of 10 (90%) controlsdeveloped viremia after challenge, while only eight out of 20 (40%)vaccinates had transient viremia, or viremia lasting only a few days atmost. Importantly, no WNV disease clinical signs were observed in any ofthe challenged vaccinated animals throughout the observation period.(Transient febrile responses were observed in one control and twovaccinated horses. However, there was no evidence to suggest theseresponses were due to WNV infection.) Petechial hemorrhage in whitematter and subdural hemorrhage were found in the brain tissue from onecontrol animal. WNV was isolated from the brain but not from CSF andspinal cord samples collected from this animal. No WNV was isolated fromany of the tissue samples collected from other challenged horses.

[0060] Results from this study demonstrated a significant protectionagainst both viremia and signs of clinical WNV disease in horsesvaccinated with the test combination vaccine.

[0061] A second study has conducted with a protocol similar to thatabove, except that a monovalent WNV vaccine (WNV vaccine alone) wasutilized, and all horses were challenged with WNV at 12 months after thesecond vaccination. Nine out of 11 (81.8%) of the controls developedviremia after challenge, while only one out of 19 (5.3%) of thevaccinates had transient viremia. No WNV associated clinical signs wereobserved in any of the challenged animals throughout the observationperiod. No febrile responses were observed in any of the challengedhorses. No WNV was isolated from any of the tissue or CSF samplescollected from any of the challenged horses. (Prior to challenge at theend of the 12 month period, 17 of the nineteen vaccinated horses hadplaque reduction neutralization test (PRNT) titers of 5 or greater,while the control group remained negative (<5).)

[0062] Results from this second study demonstrate a significantprotection (94% of preventable fraction) against viremia in horsesvaccinated with the killed monovalent WNV vaccine. These results alsodemonstrate a long duration of protective immunity.

EXAMPLE 5

[0063] Evaluation of Efficacy of DNA Test Vaccine in Horses UnderExperimental Conditions

[0064] This example demonstrates the efficacy of a West Nile Virus (WNV)DNA vaccine, as part of a further embodiment of the invention. The DNAvaccine contained 100 μg of purified DNA adjuvanted with 5% SP oil per 2mL dose, and was evaluated against experimental WNV challenge.

[0065] For the composition of the WNV DNA vaccine, bacterial cells wereharvested from a culture passaged 10 times from a master seed using E.coli DH₁₀B obtained from Invitrogen (Carlsbad, Calif.) containing a WestNile plasmid pCBWN obtained from the Centers for Disease Control (FortCollins, Colo.). The bacterial cells were suspended in glucose-tris-EDTAbuffer and lysed with sodium hydroxide and sodium dodecyl sulfate. Thelysate was neutralized with a potassium acetate solution. Theprecipitated complex material containing DNA, RNA, cell debris andproteins was removed by filtration. The filtrate was precipitated withthe addition of isopropyl alcohol. The precipitate was collected bycentrifugation and resuspended in buffer. This process was repeatedusing ammonium acetate. The precipitate collected was resuspended inbuffer and loaded into a chromatography column packed with Polyflo®)resin. The column was then washed and the plasmid DNA was eluted fromthe column. The eluate was finally diafiltered extensively againstphosphate buffer saline. The purified plasmid DNA stocks were thenshipped for blending. The test vaccine contained 100 μg of plasmid DNAadjuvanted with 5% SP oil.

[0066] The horses used for testing were randomly assigned into twogroups: 20 animals received the WNV DNA vaccine, and 10 animals wereused as controls. The first group were vaccinated intramuscularly withtwo 2.0 mL doses of vaccine three weeks apart. The control horsesreceived no vaccinations or placebos. One group of horses (9 vaccinatesand 5 controls) were challenged 5 weeks after the second vaccination,whereas a second group of horses (11 vaccinates and 5 controls) werechallenged 12 weeks after the second vaccination. Briefly, Aedesalbopictus mosquitoes which had been infected with WNV 12 days prior tothe horse challenge, were allowed to feed on each horse for at least 5minutes. Following challenge, only those mosquitoes which were found tohave engorged with blood meals from each horse were frozen at −20° C.,and the virus load was titrated as a pool subsequently. Mosquitoes werethen homogenized by vortexing using diluent. The homogenate wascentrifuged and the supernatant was removed for titration on Vero cells.

[0067] After challenge, rectal temperatures were taken twice daily forat least 9 days and clinical signs were monitored twice a day for atleast 21 days. Serum samples were taken twice a day for the first 9 dayspost challenge (DPC); once daily from 10 to 14 DPC and finally at 21DPC. Virus isolation was performed on serum samples from 0 DPC to 10 DPCfor the first challenge group and from 0 DPC to 11 DPC for the secondchallenge group. The first group of 14 horses and the second group of 16horses were euthanized and necropsied 28 to 37 DPC and 29 to 38 DPC,respectively. Cerebrospinal fluid (CSF) and tissue samples from thecerebrum, cerebellum and the brain stem were collected for grosspathology and virus isolation.

[0068] At 14 days post second vaccination, 6 of 20 vaccinated had ameasurable titer of 2 or greater, and one horse had a titer of 5.Vaccination had conferred protection against experimental West Nilechallenge using mosquitoes. Viremia was detected in 5 of 5 controlanimals and 4 of 9 vaccinates in the first challenge group, whereas 4 of5 controls and 2 of 11 vaccinates were viremic in the second challengegroup of horses. The viremia detected was transient, and occurred onlywithin the first six days after challenge. As a whole, viremia wasdetected in 9 out of 10 (90%) control horses, while only 6 out of 20(30%) vaccinates were detected with viremia.

[0069] No neurological signs attributable to WNV infection were observedin any of the study horses throughout the challenge observation period.No WNV was isolated from any of the tissue samples collected from any ofthe study horses. One horse from the first challenge group which waseuthanized on 7 DPC showed no gross or microscopic evidence ofencephalitis or meningitis.

[0070] Results from this study demonstrated a significant protectionagainst viremia in horses vaccinated with the test vaccine.

What is claimed is:
 1. A safened vaccine composition which comprises: aneffective immunizing amount of an immunogenically active componentselected from the group consisting of an inactivated whole or subunitWest Nile virus, an antigen derived from said virus, DNA derived fromsaid virus, and a mixture thereof; an immunogenically stimulating amountof a metabolizable oil; and a pharmacologically acceptable carrier. 2.The composition according to claim 1 wherein the immunogenically activecomponent is an inactivated whole or subunit West Nile virus, andfurther wherein a dosage unit of said vaccine comprises about 1×10⁴TCID₅₀ to about 1×10⁸ TCID₅₀ of said virus.
 3. The composition accordingto claim 1 wherein the metabolizable oil is SP oil.
 4. The compositionaccording to claim 2 wherein the immunogenically active component is aninactivated whole West Nile virus.
 5. The composition according to claim3 wherein said oil is present in the amount of about 4% to 10% vol/vol.6. The composition according to claim 4 wherein said virus is present insufficient quantity to provide at least about 1×10⁴ TCID₅₀ per unit doseof said composition.
 7. The composition according to claim 5 whereinsaid oil is present in the amount of about 5% vol/vol.
 8. Thecomposition according to claim 7 wherein said virus is present insufficient quantity to provide at least about 1×10⁶ TCID₅₀ per unitdose.
 9. The composition according to claim 8 wherein said virus isinactivated whole West Nile virus.
 10. A method for the prevention oramelioration of West Nile encephalitis in equidae which comprisesadministering to said equidae a safened vaccine composition whichcomprises an effective immunizing amount of an immunogenically activecomponent selected from the group consisting of an inactivated whole orsubunit West Nile virus, an antigen derived from said virus, DNA derivedfrom said virus, and a mixture thereof; an immunogenically stimulatingamount of a metabolizable oil; and a pharmacologically acceptablecarrier.
 11. The method according to claim 10 having said vaccinecomposition wherein the immunogenically active component is aninactivated whole or subunit West Nile virus.
 12. The method accordingto claim 10 having said vaccine composition wherein the metabolizableoil is SP oil.
 13. The method according to claim 10 wherein said equidaeare horses.
 14. The method according to claim 13 wherein said horses arepregnant mares.
 15. The method according to claim 10 wherein saidvaccine composition is administered parenterally.
 16. The methodaccording to claim 10 wherein said vaccine composition is administeredintramuscularly.
 17. The method according to claim 12 having a vaccinecomposition wherein the immunogenically active component is inactivatedwhole West Nile virus.
 18. The method according to claim 17 wherein saidvirus is present in an amount sufficient to provide at least about 1×10⁴TCID₅₀ and up to about 1×10⁹ TCID₅₀ per unit dose.
 19. The methodaccording to claim 18 wherein said virus is present in an amountsufficient to provide at least about 1×10⁶ TCID₅₀ per unit dose, and atleast two doses are administered to each said equidae.
 20. The methodaccording to claim 19 wherein the metabolizable oil is SP oil and ispresent in an amount of about 5% vol/vol.
 21. The composition accordingto claim 6 further comprising another vaccine component directed againstrabies virus, Eastern equine encephalitis virus, Western equineencephalitis virus, Venezuelan equine encephalitis virus, equine herpesvirus such as EHV-1 or EHV-4, Ehrlichia risticii, Streptococcus equi,tetanus toxoid, equine influenza virus (EIV).
 22. A vaccine composition,comprising: a) at least about 1×10⁴ TCID₅₀ and up to about 1×10⁹ TCID₅₀per unit dose of inactivated West Nile virus; and b) about 4% to 10%vol/vol of a metabolizable oil adjuvant comprising about 1 to 3%polyoxyethylene-polyoxypropylene block copolymer, about 2 to 6% ofsqualane and about 0.1 to 0.5% of polyoxyethylene sorbitan monooleate.23. A safened and effective West Nile virus vaccine for equidae,comprising: a) at least about 1×10⁶ TCID₅₀ to about 1×10⁸ TCID₅₀ perunit dose of killed or inactivated West Nile virus, and b) at leastabout 1% vol/vol of an adjuvant comprising at least one metabolizableoil and at least one wetting or dispersing agent.
 24. The vaccine ofclaim 23, wherein said vaccine comprises at least about 1×10⁷ TCID₅₀ ofsaid virus.
 25. The vaccine of claim 24, wherein said vaccine comprisesat least about 5×10⁷ TCID₅₀ of said virus.
 26. The vaccine of claim 23,wherein said vaccine is formulated into two dosage units.
 27. Thevaccine of claim 23, comprising at least about 4% of said adjuvant. 28.The vaccine of claim 27, comprising about 4% to 10% of said adjuvant.29. The vaccine of claim 23, wherein said adjuvant is SP oil.
 30. Thevaccine of claim 23, comprising at least two wetting or dispersingagents.
 31. The vaccine of claim 30, wherein said wetting or dispersingagents are selected from the group consisting of non-ionic surfactants.32. The vaccine of claim 31, wherein said non-ionic surfactants areselected from the group consisting of polyoxyethylene/polyoxypropyleneblock copolymers and polyoxyethylene esters.
 33. A vaccine regimen forhorses or other equidae, comprising two dosage units of killed orinactivated West Nile virus, wherein each said dosage unit comprisesabout 0.5 to 5 milliliters of a composition containing at least about5×10⁷ TCID₅₀ of said virus and about 1 to 10% vol/vol of an adjuvant,said adjuvant comprising at least one metabolizable oil and at least twononionic surfactants, and further wherein said dosage unit comprises apharmacologically acceptable carrier.
 34. The vaccine regimen of claim33, wherein said adjuvant is SP oil.
 35. The vaccine regimen of claim34, wherein said SP oil comprises squalane in an amount of about 2 to 6%by weight of said adjuvant, about 1 to 3% ofpolyoxyethylene/polyoxypropylene block copolymer by weight of saidadjuvant, and about 0.1 to 0.5% of polyoxyethylene sorbitan monooleateby weight of said adjuvant.
 36. The vaccine regimen of claim 33, furthercomprising a vaccine component directed to at least one member selectedfrom the group consisting of influenza virus, Eastern, Western andVenezuelan rhinopneumonitis virus and tetanus toxoid.
 37. A West Nilevirus vaccine composition comprising West Nile plasmid DNA in an amountof about 50 to 3,000 micrograms per dose.
 38. The composition of claim37, wherein said composition comprises about 100 to 1,000 micrograms perdose.
 39. The composition of claim 38, wherein said compositioncomprises about 100 to 250 micrograms per dose.
 40. A West Nile virusvaccine regimen which comprises administering to a horse at least aboutone dose of the composition of claim
 37. 41. The regimen of claim 40,which comprises administering at least about two doses.